Purification and characterization of adenosine receptors have been continued. Using a novel affinity chromatography system which we had previously developed, A1 adenosine receptors were successfully purified to homogeneity, not only from rat brain membranes but also from rat testicular membranes and human brain membranes. It was shown that the molecular weight of these purified receptors were similar (34,000-41,000) and that all these receptors are glycosylated. Immunological properties among these three A1 adenosine receptors were compared by a western blot using a polyclonal antibody against the purified rat brain A1 adenosine receptor. Immunocytochemical studies of the distribution of A1 adenosine receptors in the rat brain were also performed. Discrete puncta were observed in the cerebral cortex, supraoptic and paraventricular nuclei, hippocampus, purkinje cells of the cerebellum, red nucleus and raphe magnus.